Preclinical assessment of pan-influenza A virus CRISPR RNA therapeutics in a human lung alveolus chip

CRISPR technology offers an entirely new approach to therapeutic development because it can target specific nucleotide sequences with high specificity, however, preclinical animal models are not useful for evaluation of their efficacy and potential off-target effects because of high gene sequence variations between animals and humans. Here, we explored the potential of using the CRISPR effector Cas13 to develop a new therapeutic approach for influenza A virus (IAV) infections based on its ability to specifically and robustly cleave single-strand viral RNA using a complementary CRISPR RNA (crRNA). We engineered crRNAs to target highly conserved regions in the IAV genome to create a potential pan-viral treatment strategy. A human lung alveolus chip (Lung Chip) lined by human primary alveolar epithelial cells interfaced with human primary pulmonary microvascular endothelial cells and infected with a pandemic IAV H3N2 strain was used to evaluate the on-target and off-target effects of these antiviral crRNA therapeutics. Our data show that the crRNAs targeting highly conserved regions in the IAV genome potently reduced viral replication in the alveolar airspace in the Lung Chip, and this was accompanied by suppression of the human host inflammatory response as indicated by a significant reduction in cytokine production and recruitment of immune cells. Importantly, only minimal off-target effects were observed based on transcriptomic analyses. As these crRNAs inhibit replication of influenza H1N1 and H3N2 in A549 cells as well as H3N2 in Lung Chips, these findings support use of CRISPR-Cas13 as a potentially viable approach to develop pan-IAV therapeutics for combating future influenza pandemics. The results also demonstrate that human Organ Chips be useful as more clinically relevant preclinical models for testing the efficacy and safety of crRNA therapeutics.